Pyrazinone Modulator of Corticotropin-Releasing Factor Receptor Activity

ABSTRACT

The invention relates to the compound (S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile, pharmaceutical compositions of the compound, and methods of using the compound for the treatment of psychiatric disorders and neurological diseases including depression, anxiety related disorders, irritable bowel syndrome, addiction and negative aspects of drug and alcohol withdrawal, and other conditions associated with CRF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 61/085,230 filed Jul. 31, 2008.

BACKGROUND OF THE INVENTION

The invention relates to the compound(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile,pharmaceutical compositions of the compound, and methods of using thecompound for the treatment of psychiatric disorders and neurologicaldiseases including depression, anxiety related disorders, irritablebowel syndrome, addiction and negative aspects of drug and alcoholwithdrawal, and other conditions associated with CRF.

Corticotropin releasing factor (CRF), a 41 amino acid peptide, is theprimary physiological regulator of proopiomelanocortin (POMC) derivedpeptide secretion from the anterior pituitary gland [Rivier, J. et al.,Proc. Nat. Acad. Sci. (USA) 80: 4851 (1983); Vale, W. et al., Science213: 1394 (1981)]. In addition to its endocrine role at the pituitarygland, immunohistochemical localization of CRF has demonstrated that thehormone has a broad extrahypothalamic distribution in the centralnervous system and produces a wide spectrum of autonomic,electrophysiological and behavioral effects consistent with aneurotransmitter or neuromodulator role in brain [Vale, W. et al., Rec.Prog. Horm. Res. 39: 245 (1983); Koob, G. F. Persp. Behav. Med. 2: 39(1985); De Souza, E. B. et al., J. Neurosci. 5: 3189 (1985)]. There isevidence that CRF plays a significant role in integrating the responseof the immune system to physiological, psychological, and immunologicalstressors [Blalock, J. E. Physiological Reviews 69: 1 (1989); Morley, J.E. Life Sci. 41: 527 (1987)].

Over-expression or under-expression of CRF has been proposed as anunderlying cause for several medical disorders. Such treatable disordersinclude affective disorder, anxiety, depression, headache, irritablebowel syndrome, post-traumatic stress disorder, supranuclear palsy,immune suppression, Alzheimer's disease, gastrointestinal diseases,anorexia nervosa or other feeding disorders, drug addiction, drug oralcohol withdrawal symptoms, inflammatory diseases, cardiovascular orheart-related diseases, fertility problems, human immunodeficiency virusinfections, hemorrhagic stress, obesity, infertility, head and spinalcord traumas, epilepsy, stroke, ulcers, amyotrophic lateral sclerosis,hypoglycemia, hypertension, tachycardia and congestive heart failure,stroke, osteoporosis, premature birth, psychosocial dwarfism,stress-induced fever, ulcer, diarrhea, post-operative ileus and colonichypersensitivity associated with psychopathological disturbance andstress [for reviews see McCarthy, J. R.; Heinrichs, S. C.; Grigoriadis,D. E. Cur. Pharm. Res. 5: 289-315 (1999); Gilligan, P. J.; Robertson, D.W.; Zaczek, R. J. Med. Chem. 43: 1641-1660 (2000), Chrousos, G. P. Int.J. Obesity, 24, Suppl. 2, S50-S55 (2000); Webster, E.; Torpy, D. J.;Elenkov, I. J.; Chrousos, G. P. Ann. N. Y. Acad. Sci. 840: 21-32 (1998);Newport, D. J.; Nemeroff, C. B. Curr. Opin. Neurobiology, 10: 211-218(2000); Mastorakos, G.; Ilias, I. Ann, N.Y. Acad. Sci. 900: 95-106(2000); Owens, M. J.; Nemeroff, C. B. Expert Opin. Invest. Drugs 8:1849-1858 (1999); Koob, G. F. Ann. N.Y. Acad. Sci., 909: 170-185(2000)].

There is evidence that CRF plays a role in affective disorders includingdepression (for example, major depression, single episode depression,recurrent depression, child abuse induced depression, and postpartumdepression), dysthemia, bipolar disorders, and cyclothymia. Inindividuals afflicted with major depression, the concentration of CRF issignificantly increased in the cerebrospinal fluid (CSF) of drug-freeindividuals [Nemeroff, C. B. et al., Science 226: 1342 (1984); Banki, C.M. et al., Am. J. Psychiatry 144: 873 (1987); France, R. D. et al.,Biol. Psychiatry 28: 86 (1988); Arato, M. et al., Biol Psychiatry 25:355 (1989)]. Furthermore, the density of CRF receptors is significantlydecreased in the frontal cortex of suicide victims, consistent with ahypersecretion of CRF [Nemeroff, C. B. et al., Arch. Gen. Psychiatry 45:577 (1988)]. In addition, there is a blunted adrenocorticotropin (ACTH)response to CRF (i.v. administered) observed in depressed patients[Gold, P. W. et al., Am J. Psychiatry 141: 619 (1984); Holsboer, F. etal., Psychoneuroendocrinology 9: 147 (1984); Gold, P. W. et al, New Eng.J. Med. 314: 1129 (1986)]. Preclinical studies in rats and non-humanprimates provide additional support for the hypothesis thathypersecretion of CRF may be involved in the symptoms seen in humandepression [Sapolsky, R. M. Arch. Gen. Psychiatry 46: 1047 (1989)].There is preliminary evidence that tricyclic antidepressants can alterCRF levels and thus modulate the numbers of CRF receptors in brain[Grigoriadis et al., Neuropsychopharmacology 2: 53 (1989)].

There is evidence that CRF plays a role in the etiology of anxiety andrelated disorders including anxiety with co-morbid depressive illness,panic disorder, phobic disorders, social anxiety disorder,obsessive-compulsive disorder, post-traumatic stress disorder, andatypical anxiety disorders [The Merck Manual of Diagnosis and Therapy,16^(th) edition (1992)]. Emotional stress is often a precipitatingfactor in anxiety disorders, and such disorders generally respond tomedications that lower response to stress. Excessive levels of CRF areknown to produce anxiogenic effects in animal models [see Britton, D. R.et al., Life Sci. 31: 363 (1982); Berridge, C. W., Dunn, A. J. Regul.Peptides 16: 83 (1986); Berridge, C. W.; Dunn, A. J. Horm. Behav. 21:393 (1987)]. CRF produces anxiogenic effects in animals and interactionsbetween benzodiazepine/non-benzodiazepine anxiolytics and CRF have beendemonstrated in a variety of behavioral anxiety models [Britton, D. R.et al., Life Sci. 31: 363 (1982); Berridge, C. W., Dunn, A. J. Regul.Peptides 16: 83 (1986)]. Preliminary studies using the putative CRFreceptor antagonist α-helical ovine CRF (9-41) in a variety ofbehavioral paradigms demonstrate that the antagonist produces“anxiolytic-like” effects that are qualitatively similar to thebenzodiazepines [Berridge, C. W.; Dunn, A. J. Horm. Behav. 21: 393(1987), Dunn, A. J.; Berridge, C. W. Brain Research Reviews 15: 71(1990)].

Neurochemical, endocrine, and receptor binding studies have all Sdemonstrated interactions between CRF and benzodiazepine anxiolytics,providing further evidence for the involvement of CRF in thesedisorders. Chlordiazepoxide attenuates the “anxiogenic” effects of CRFin both the conflict test [Britton, K. T. et al. Psychopharmacology 86:170 (1985); Britton, K. T. et al. Psychopharmacology 94: 306 (1988)] andin the acoustic startle test [Swerdlow, N. R. et al. Psychopharmacology88: 147 (1986)] in rats. The benzodiazepine receptor antagonist(Ro15-1788), which was without behavioral activity alone in the operantconflict test, reversed the effects of CRF in a dose-dependent mannerwhile the benzodiazepine inverse agonist (FG7142) enhanced the actionsof CRF [Britton, K. T. et al. Psychopharmacology 94: 306 (1988)]. Ofparticular interest is that preliminary studies examining the effects ofa CRF receptor antagonist (α-helical CRF9-41) in a variety of behavioralparadigms have demonstrated that the CRF antagonist produces“anxiolytic-like” effects qualitatively similar to the benzodiazepines[for review see G. F. Koob and K. T. Britton, In:Corticotropin-Releasing Factor: Basic and Clinical Studies of aNeuropeptide, E. B. De Souza and C. B. Nemeroff eds., CRC Press p 221(1990)].

In addition to modulating the HPA-axis, CRF is considered to be a keymodulator of the gut-brain axis. Evidence exists indicating that CRF mayplay a role in mediating stress-related gastrointestinal disorders[Gabry, K. E. et al. Molecular Psychiatry 7(5): 474-483 (2002)] such asirritable bowel syndrome (IBS), post-operative ileus, and colonichypersensitivity associated with psychopathological disturbance andstress [for reviews see E. D. DeSouza, C. B. Nemeroff, Editors;Corticotropin-Releasing Factor: Basic and Clinical Studies of aNeuropeptide, E. B. De Souza and C. B. Nemeroff eds., CRC Press p 221(1990) and Maillot C. et al. Gastroenterology, 119: 1569-1579 (2000);Fukudo, S. J. Gastroenterol, 42 (Suppl XVII): 48 (2007); Taché, Y.;Bonaz, B. J. Clin. Invest. 117: 33 (2007)]. In rats it has beendemonstrated that i.p. administration of CRF₁ antagonist JTC-017 blockedan increase in fecal output induced by exposure to chronic colorectaldistention [Saito, K. et al. Gastroenterol., 129: 1533 (2005)].Additionally, JTC-017 attenuated the anxiety-related behavior seen afterexposure to acute colorectal distention. CRF-stimulated colonic motilityin rats was also attenuated by central administration of CRF_(1/2)peptide antagonist astressin [Tsukamoto, K. et al. Am. J. Physiol.Regul. Integr. Comp. Physiol. 290: RI 537 (2006)]. In healthy humans,i.v. administration of CRF was shown to affect rectal hypersensitivityand mimic a stress-induced visceral response specific to IBS patients[Nozu, T.; Kudaira, M. J. Gastroenterol. 41: 740 (2006)]. These datasuggest that CRF antagonists may be useful for the treatment of IBS.

Antagonists of CRF₁ have been examined for use as treatments foraddiction and the negative aspects of drug withdrawal [Steckler, T.;Dautzenberg, F. M. CNS Neurol. Disord. Drug Targets 5: 147 (2006)].Withdrawal from nicotine, cocaine, opiates, and alcohol often leads to anegative emotional state and elevated levels of anxiety. Theseundesirable effects can sometimes be counteracted by increasing selfadministration of the substance, which leads to relapse to the addictedstate. External stressors can often lead to a relapse in abuse as well.

CRF receptor antagonists may be useful for treatment of the negativeaffective aspects of withdrawal from nicotine. Pretreatment of nicotinedependent rats with CRF_(1/2) peptide antagonist D-Phe CRF₍₁₂₋₄₁₎ wasshown to prevent the elevations in brain reward threshold associatedwith nicotine withdrawal [Bruijnzeel, A. W. et al. Neuropsychopharmacol.32: 955 (2007)]. D-Phe CRF₍₁₂₋₄₁₎ also caused a decrease instress-induced reinstatement of nicotine-seeking behavior in rats[Zislis, G. et al. Neuropharmacol. 53: 958 (2007)]. Additionally, anincrease in nicotine intake after a period of abstinence, often seenwith nicotine dependence, could be blocked in rats by pretreatment withthe CRF₁ antagonist MPZP [Specio, S. E. et al. Psychopharmacol. 196: 473(2008); George, O. et al. Proc. Natl. Acad. Sci. U.S.A. 104: 17198(2007)].

Evidence from animal studies also suggests that the effects of cocaineand morphine withdrawal and relapse may be attenuated by antagonism ofthe CRF receptor. The CRF₁ antagonist CP-154,526 was shown to attenuatespiradoline-induced reinstatement of cocaine seeking behavior insquirrel monkeys [Valdez, G. R. et al. J. Pharm. Exp. Ther. 323: 525(2007)] as well as cue-induced reinstatement of methamphetamine-seekingbehavior in rats [Moffett, M. C. et al. Psychopharmacol. 190: 171(2007)]. Lorazepam dependent rats pretreated with CRF₁ antagonistR121919 [Holsboer, F. et al. Eur. J. Pharmacol. 583: 350 (2008)] beforeprecipitation of withdrawal showed reduced HPA axis activation andreduced anxiety behaviors in the defensive withdrawal model [Skelton, K.H. et al. Psychopharmacol. 192: 385 (2007)]. R121919 was similarly ableto attenuate the severity of precipitated morphine withdrawal andwithdrawal-induced HPA axis activation [Skelton, K. H. et al. Eur. J.Pharmacol. 571: 17 (2007)]. The amount of opiate exposure duringself-administration as well as the length of abstinence can affectrelapse. Rats allowed to self-administer cocaine for longer periods oftime (6 h daily) were more susceptible to reinstatement by cocaine,electric foot shock, or administered CRF than those allowed toself-administer for shorter periods (2 h daily) [Mantsch, J. R. et al.Psychopharmacol. 195: 591 (2008)]. In another study, CRF₁ antagonistsMPZP and antalarmin were shown to reduce cocaine self-administration inrats with extended daily cocaine access [Specio, S. E. Psychopharmacol.196: 473 (2008)].

There is evidence suggesting that CRF₁ antagonists may help block thenegative emotional aspects, excessive alcohol drinking, andstress-induced relapse seen in ethanol dependence [Heilig, M. et al.Trends Neurosci. 30: 399 (2007)]. Ethanol-dependent wild-type mice showan increase in ethanol self-administration during withdrawal, but onlyafter a period of abstinence [Chu, K. G. F. Pharmacol. Biochem. Behav.86: 813 (2007)]. This effect was reversed by administration of the CRF₁antagonist antalarmin. CRF₁ knockout (KO) mice do not show this tendencytoward increased self-administration. When treated with CRY, antagonistsR121919 or antalarmin, ethanol-dependent rats showed a reduction inexcessive ethanol self-administration during acute withdrawal [Funk, C.K. et al. Biol. Psychiatry 61: 78 (2007)]. Non-dependent rats treatedwith these CRF₁ antagonists, however, showed no effect on ethanolself-administration. Similarly, CRF₁ antagonist MPZP selectively reducedexcessive ethanol self-administration during acute withdrawal independent rats [Richardson, H. N. et al. Pharmacol. Biochem. Behav. 88:497 (2008)]. In another study, a novel CRF₁ antagonist selectivelyreduced excessive ethanol self-administration induced by stress independent rats [Gehlert, D. R. et al. J. Neurosci. 27: 2718 (2007)].These studies demonstrate that antagonism of CRF₁ receptors canselectively block excessive ethanol self-administration withoutaffecting basal self-administration levels. This suggests that CRF₁antagonists could be useful for the treatment of alcohol dependence.

It has been further postulated that CRF has a role in cardiovascular orheart-related diseases arising from stress such as hypertension,tachycardia and congestive heart failure, stroke (methods for using CRF₁antagonists to treat congestive heart failure are described in U.S.patent application Ser. No. 09/248,073, filed Feb. 10, 1999, now U.S.Pat. No. 6,043,260 (Mar. 28, 2000).

It has also been suggested that CRF₁ antagonists are useful for treatingarthritis and inflammation disorders [Webster, E. L. et al. J.Rheumatol. 29(6): 1252-61 (2002); Murphy, E. P. et al. Arthritis Rheum.44(4): 782-93 (2001)].

It has also been suggested that CRF₁ antagonists are useful for skindisorders [Zouboulis, C. C. et al. Proc. Natl. Acad. Sci. 99: 7148-7153(2002)]. Stress-induced exacerbation of chronic contact dermatitis wasblocked by a selective CRF₁ antagonist in an animal model, suggestingthat CRF₁ is involved in the stress-induced exacerbation of chroniccontact dermatitis and that CRF₁ antagonist may be useful for treatingthis disorder [Kaneko, K. et al. Exp. Dermatol, 12(1): 47-52 (2003)].

Studies have demonstrated that CRF₁ antagonists may be useful as hairgrowth stimulators (WO2002/19975 discloses cell culture assays for theuse of CRF antagonists in stimulating KBM-2 cell production). Thus, CRFantagonists may be useful in treatment of hair loss.

DESCRIPTION OF THE INVENTION

The invention encompasses(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile(Compound I) and pharmaceutical compositions and methods for modulatingCRF in patients with medical conditions associated with aberrant levelsof CRF.

The invention includes all pharmaceutically acceptable salt forms of thecompounds. Pharmaceutically acceptable salts are those in which thecounter ions do not contribute significantly to the physiologicalactivity or toxicity of the compounds and as such function aspharmacological equivalents. These salts can be made according to commonorganic techniques employing commercially available reagents. Someanionic salt forms include acetate, acistrate, besylate, bromide,chloride, citrate, fumarate, glucouronate, hydrobromide, hydrochloride,hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate,phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate. Somecationic salt forms include ammonium, aluminum, benzathine, bismuth,calcium, choline, diethylamine, diethanolamine, lithium, magnesium,meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium,tromethamine, and zinc.

As Compound I possesses an asymmetric carbon atom, the inventionincludes the racemate as well as the individual enantiometric forms ofCompound I and chiral and racemic intermediates as described herein. Theuse of a single designation such as (R) or (S) is intended to includemostly one stereoisomer. Mixtures of isomers can be separated intoindividual isomers according to methods known in the art.

“Treatment,” “therapy,” “regimen,” and related terms are used asunderstood by medical practitioners in the art.

“Therapeutically effective” means the amount of agent required toprovide a meaningful patient benefit as understood by medicalpractitioners in the art. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

“Patient” means a person suitable for therapy as understood by medicalpractitioners in the art.

Synthetic Methods

Compound I can be prepared by methods known in the art including thosedescribed in Schemes 1-8, Reasonable variations of the describedprocedures, together with synthetic methods which would be evident toone skilled in the art, are intended to be within the scope of thepresent invention.

Abbreviations used in the schemes generally follow conventions used inthe art. Chemical abbreviations used in the specification and examplesare defined as follows: “NaHMDS” for sodium bis(trimethylsilyl)amide;“DMF” for N,N-dimethylformamide; “MeOH” for methanol; “NBS” forN-bromosuccinimide; “Ar” for aryl; “TFA” for trifluoroacetic acid; “LAH”for lithium aluminum hydride; “BOC”, “DMSO” for dimethylsulfoxide; “h”for hours; “rt” for room temperature or retention time (context willdictate); “min” for minutes; “EtOAc” for ethyl acetate; “THF” fortetrahydrofuran; “EDTA” for ethylenediaminetetraacetic acid; “Et₂O” fordiethyl ether; “DMAP” for 4-dimethylaminopyridine; “DCE” for1,2-dichloroethane; “ACN” for acetonitrile; “DME” for1,2-dimethoxyethane; “HOBt” for 1-hydroxybenzotriazole hydrate; “DIEA”for diisopropylethylamine, “Nf” for CF₃(CF₂)₃SO₂—; and “TMOF” fortrimethylorthoformate.

Two routes for the synthesis of the (S)-amine (9) are shown in Schemes 1and 2. In Route A (Scheme 1), the (S)-chiral center in the amine can beestablished by a Strecker synthesis involving diastereoselectiveaddition of cyanide to the chiral imine 4 (Bayston, D. J.; Griffin, J.L. W.; Gruman, A.; Polywka, M. E. C.; Scott, R. M. U.S. Pat. No.6,191,306). Imine 4 in turn can be derived from the commerciallyavailable chiral amine 3. In Route B, the chiral amine can be obtainedfrom racemic intermediate 13 by chiral chromatographic separation.

Route A starts with condensation of commercially available cyclopropanecarboxaldehyde 2 and (S)-1-phenethyl amine 3 and can provide the chiralimine 4. Treatment in situ with potassium cyanide, followed byhydrolysis of the intermediate α-amino nitrile 5 under acidic conditionscan generate acid 6. Borane reduction of 6 can afford alcohol 7, whichcan be methylated by treatment with sodium hydride and methyl iodide toprovide the ether 8 in 76% yield. Reductive cleavage of the benzyl groupcan generate the key chiral amine 9.

Route B to (S)-amine 9 begins with conversion of commercially availableα-methoxy acid 10 to amide 11, followed by addition of cyclopropylGrignard which can afford ketone 12. Reductive animation, followed byprotection of the amine as the (benzyloxy)carbamate, can provide racemicintermediate 13. Separation of the enantiomers by chromatography cangenerate the single enantiomer 14. Deprotection under acidic conditionscan provide (S)-amine 9.

Synthesis of the dibromopyrazinone 16 and dichloropyrazinone 17 isoutlined in Scheme 3. Alkylation of amine 9 with chloroacetonitrile canprovide α-amino nitrile 15. Dibromopyrazinone ring formation can beaffected by treatment of the nitrile with excess oxalyl bromide toprovide 16. Alternatively, treatment of the nitrile 15 with oxalylchloride can provide dichloropyrazinone 17 [Vekemans, J.;Pollers-Wieërs, C.; Hoornaert, G. J. Heterocycl. Chem. 20: 919-923(1983)].

Synthesis of the pyridyl amine fragment 26 is shown in Schemes 4 and 5.Commercially available 2-amino-3-methylpyridine 18 can be nitrated bytreatment with nitric acid and sulfuric acid using a modification of thetwo-step, one pot procedure of Hawkins and Roe [Hawkins, G3. F.; Roe, A.J. Org. Chem. 14: 328-332 (1949)] to provide pyridone 19. Conversion tomethoxypyridine 20 can be accomplished in high yield in a two-stepprocedure starting with treatment of 19 with POCl₃ followed bymethanolysis. Installation of the 6-methyl substituent can be achievedthrough two different procedures, both involving vicarious nucleophilicaromatic substitution at the 6-position of nitropyridine 20. Method Ainvolves generation of the anion of t-butyl chloroacetate, followed byhydrolysis of the resulting ester 21 to give the acid 22.Decarboxylation can be affected by heating under basic conditions in DMFto provide the 6-methylpyridine 23. Alternatively in Method B, the6-methyl group can be installed directly by treatment of 20 withtrimethylsulfoxonium iodide.

Completion of the synthesis of pyridyl amine 26 can be commenced byheating 23 under acidic conditions for 1-2 hours to furnish pyridone 24,The difluoromethyl ether 25 can be prepared by selective O-alkylation of24 with difluorocarbene using, for example, the silyl ester of2-fluorosulfonyldifluoroacetate (Dolbier, W. R.; Tian, F.; Duan, J. X.;Chen, Q. Y. Org. Synth. 2003, 80, 172-176; Dolbier, W. R.; Tian, F.;Duan, J. X.; Li, A.; Ait-Mohand, S.; Bautista, O.; Buathong, S.; Baker,J. M.; Crawford, J.; Anselme, P.; Cai, X. H.; Modzelewska, A.; Koroniak,H.; Battiste, M, A.; Chen, Q. Y. J. Fluorine Chem. 2004, 125, 459-469;Cai, X.; Zhai, Y.; Ghiviriga, I.; Abboud, K. A.; Dolbier, W. R. J. Org.Chem. 2004, 69, 4210-4215) or 2-fluorosulfonyldifluoroacetic acid (Chen,Q. Y.; Wu, S. W. J. Fluorine Chem. 1989, 44, 433-440). Final conversionto the pyridyl amine 26 can be accomplished by hydrogenation.

Completion of the synthesis of 1 is shown in Scheme 6. Coupling of thedibromopyrazinone 16 with pyridyl amine 26 can be achieved in thepresence of NaHMDS to provide compound 26. Palladium-mediated couplingof 27 with zinc cyanide in DMF can provide cyanopyrazinone 1 [Maligres,P. E.; Waters, M. S.; Fleitz, F.; Askin, D. Tetrahedron Lett. 40;8193-8195 (1999)].

Synthesis of cyanopyrazinone 1 from chloropyrazinone 28 can also beeffected using zinc cyanide and palladium catalysis.

Alternatively, chloropyrazinone 29 can be converted to cyanopyrazinone 1with zinc cyanide under palladium catalysis provided the5-cyanopyrazinone 30. Demethylation of the methoxypyridine moiety can beeffected by treatment with potassium iodide in acetic acid. Subsequentselective O-alkylation of the pyridinone with the trimethylsilyl esterof 2-fluorosulfonyldifluoroacetic acid can provide cyanopyrazinone 1.

Biological Methods

CRF₁ Binding Assay. Frozen rat frontal cortex was thawed rapidly inassay buffer containing 50 mM Hepes (pH 7.0 at 23° C.), 10 mM MgCl₂, 2mM EGTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A,0.005% Triton X-100, 10 U/ml bacitracin and 0.1% ovalbumin andhomogenized. The suspension was centrifuged at 32000×g for 30 minutes.The resulting supernatant was discarded and the pellet resuspended byhomogenization in assay buffer and centrifuged again. The supernatantwas discarded and the pellet resuspended by homogenization in assaybuffer and frozen at −70° C. On the day of the experiment aliquots ofthe homogenate were thawed quickly and 25 μg/well added to 150 pM¹²⁵1-ovine-CRF (¹²⁵I-o-CRF) and drugs in a total volume of 100 μl assaybuffer. The assay mixture was incubated for 2 hr at 21° C. Bound andfree radioligand were then separated by rapid filtration using glassfiber filters (Whatman GF/B, pretreated with 0.3% PEI(polyethylenimine)) on a Brandel Cell Harvester. Filters were thenwashed multiple times with ice cold wash buffer (PBS w/o Ca²⁺ and Mg²⁺,0.01% Triton X-100; pH 7.0 at 23° C.). Non-specific binding was definedusing 1 μM DMP696, a CRF₁ selective antagonist [Li, Y-W. et al., CNSDrug Reviews 11: 21-52, (2005)]. Filters were then counted in a WallacWizard gamma counter. IC₅₀ values were determined in a five point (fivedrug concentrations) or ten point (ten drug concentrations) competitionassay using non-linear regression by Microsoft Excel-fit,

CRF₂ Binding Assay. Frozen porcine choroid plexus was thawed rapidly inassay buffer containing 50 mM Hepes (pH 7.0 at 23° C.), 10 mM MgCl₂, 2mM EGTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A,0.005% Triton X-100, 10 U/ml bacitracin and 0.1% ovalbumin andhomogenized. The suspension was centrifuged at 32000×g for 30 minutes.The resulting supernatant was discarded and the pellet resuspended byhomogenization in assay buffer and centrifuged again. The supernatantwas discarded and the pellet resuspended by homogenization in assaybuffer and frozen at −70° C. On the day of the experiment aliquots ofthe homogenate were thawed quickly and 10 μg/well added to 100 pM¹²⁵I-sauvagine and drugs in a total volume of 100 μl assay buffer. Theassay mixture was incubated for 2 hr at 21° C. Bound and freeradioligand were then separated by rapid filtration using glass fiberfilters (Whatman GF/B, pretreated with 0.3% PEI) on a Brandel CellHarvester. Filters were then washed multiple times with ice cold washbuffer (PBS w/o Ca²⁺ and Mg²⁺, 0.01% Triton X-100 (pH 7.0 at 23° C.)),Non-specific binding was defined using 1 μM α-helical CRF (9-41).Filters were then counted in a Wallac Wizard gamma counter. IC₅₀ valueswere determined in a five point (five drug concentrations) competitionassay using non-linear regression by Microsoft Excel-fit.

hCRF₁ Binding Assay. Membranes were prepared for binding using Y79cells, a human retinoblastoma line that natively expresses human CRF₁receptors [Hauger, R., et al., Journal of Neurochemistry 68: 2308-2316,(1997)]. Briefly, cells were grown and collected then the pellethomogenized in assay buffer containing 50 mM Hepes (H 7.0 at 23° C.), 10mM MgCl₂, 2 mM EGTA, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/mlpepstatin A, 0.005% Triton X-100, 10 U/ml bacitracin and 0.1% ovalbumin.The suspension was centrifuged at 32000×g for 30 minutes. The resultingsupernatant was discarded and the pellet resuspended by homogenizationin assay buffer and centrifuged again. The supernatant was discarded andthe pellet resuspended by homogenization in assay buffer and aliquotsfrozen at −70° C. On the day of the experiment aliquots were thawedquickly and 25 μg/well added to 150 pM ¹²⁵I-ovine-CRF and drugs in atotal volume of 100 μl assay buffer. The assay mixture was incubated for2 hr at 21° C. Bound and free radioligand were then separated by rapidfiltration using glass fiber filters (Whatman GF/B, pretreated with 0.3%PEI) on a Brandel Cell Harvester. Filters were then washed multipletimes with ice cold wash buffer (PBS w/o Ca²⁺ and Mg²⁺, 0.01% TritonX-100 (H 7.0 at 23° C.)). Non-specific binding was defined using 1 μMDMP696. Filters were then counted in a Wallac Wizard gamma counter. IC₅₀values were determined in a five point (five drug concentrations)competition assay using nonlinear regression by Microsoft Excel-fit.

Binding Results. The primary screen to determine binding potency (IC₅₀value) of(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrileat the CRF₁ receptor was a competition binding experiment utilizing¹²⁵I-o-CRF as the labeled peptide ligand and rat frontal cortex membraneas the receptor source.(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrilepotently and completely inhibited ¹²⁵I-o-CRF binding to rat frontalcortex membrane displaying an IC₅₀ of 0.86±0.04 nM (n=10) and a HillSlope of 0.94±0.03 (n=10). These results include both 5 point and 10point competition binding assays. For comparison DMP696, the wellcharacterized small molecule selective CRF₁ antagonist, was alsoexamined and displayed an IC₅₀ of 1.39±0.09 nM (n=10) and a Hill Slopeof 1.00±0.02 (n=10).(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrilealso bound with high affinity to the native CRF₁ receptors present onthe human retinoblastoma cell line Y79.(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrilepotently and completely inhibited ¹²⁵I-o-CRF binding to Y79 membranedisplaying an IC₅₀ of 1.81±0.46 nM (n=3). For comparison DMP696 was alsoexamined and displayed an IC₅₀ of 1.71±0.53 nM (n=3). In contrast to itshigh affinity for the CRF₁ receptor,(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrileshowed little or no affinity at the CRF₂ receptor expressed in porcinechoroid plexus membrane. While α-helical CRF (a truncated high affinityantagonist of CRF₁ and CRF₂ receptors) bound with high affinity to theCRF₂ receptors endogenously expressed on the porcine choroid plexusmembrane (IC₅₀=25.7±2.9 nM, n=3),(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitriledisplayed an IC₅₀>10,000 nM (n=3).

Situational Anxiety Behavioral Studies in Rats.(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrilehas been evaluated in a situational anxiety model to evaluate itsanxiolytic potential. In this model, rats are placed in a small,darkened chamber located in an unfamiliar open field. Vehicle-treatedrats spend most of the time within the chamber, consistent with aheightened state of anxiety [Takahashi L. K. et al, Behav. Neurosci.103: 648-654, (1989)]. This model is sensitive to anxiolytics, includingbenzodiazepines such as chlordiazepoxide [Yang X-M. et al, J. Pharmacol.Exp. Ther. 255: 1064-1070, (1990)]. In addition, previous CRF₁antagonists, such as DMP696 and DMP904 [McElroy J. F. et al.,Psychopharmacology 165: 86-92, (2002); Lelas S. et al., J. Pharmacol.Exp. Ther, 309: 293-302, (2004)] were effective this model. The effectsof(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrilewere compared with those of anxiolytic standards chlordiazepoxide(Librium®) and diazepam (Valium®) in this model. Male Sprague-Dawleyrats weighing 180-300 g were purchased from Charles River Laboratories(Wilmington, Mass.). The rats were housed individually in suspended wirecages in colony rooms maintained at a constant temperature (21±2° C.)and humidity (50±10%). The rooms were illuminated 12 hours per day. Therats had ad libitum access to food and water throughout the studies.Behavioral studies were conducted between 0600 and 1300 h. Animals weremaintained in accordance with the guidelines of the Animal Care and UseCommittee of the Bristol-Myers Squibb Company, the “Guide for Care andUse of Laboratory Animals” (Institute of Animal Laboratory Resources,1996), and the guidelines published in the National Institutes of HealthGuide for the Care and Use of Laboratory Animals. Research protocolswere approved by the Bristol-Myers Squibb Company Animal Care and UseCommittee. All compounds were prepared in 0.25% methylcellulose andadministered PO. Chlordiazepoxide, diazepam, and desipramine werepurchased from Sigma Chemical Company.(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrilewas dosed in the volume of 2 ml/kg. All standards were dosed in thevolume of 2 ml/kg. For all tests, data were analyzed by analysis ofvariance, followed by Dunnett's t-tests for individual comparisons. Thesignificance value was set at 0.05. The data are presented in the textand figures as mean±standard error of the mean (SEM). MaleSprague-Dawley rats were habituated to handling and dosing the daybefore testing. On the day of testing, all compounds were administeredPO by gavage 60 minutes before behavioral testing. To initiate testing,each animal was placed in a small galvanized steel cylinder (14 cmlength, 10 cm diameter), which was placed lengthwise against one wall ofan illuminated open field (106 cm length×92 cm width×50 cm height). Theopen field was illuminated by a 60-W incandescent bulb and illuminationwas titrated by a powerstat transformer to a 30-lux reading at theentrance to the cylinder. Behavior was assessed for 15 minutes by atrained observer unaware of treatment assignment. The latency of theanimal to exit the cylinder, defined by the placement of all four pawsinto the open field, and explore the open field was recorded (inseconds). If the animal did not leave the cylinder after 15 minutes thetrial was terminated and a score of 900 seconds was recorded. ThePlexiglas chamber and the cylinder were cleaned with 1.0% glacial aceticacid between animals to prevent olfactory cues from influencing thebehavior of subsequently tested animals. Following behavioral testing,animals were sacrificed and plasma samples and brains were taken foranalysis of plasma exposure and central receptor occupancy.

Situational Anxiety Behavioral Studies Results. Vehicle-treated animalsshowed long latencies to exit the dark chamber and explore the openfield. The mean exit latencies were 792±108 and 794±106 s in thechlordiazepoxide and diazepam studies, respectively (88% of the totaltest duration of 900 s for both studies). The benzodiazepine anxiolyticschlordiazepoxide and diazepam both dose-dependently decreased exitlatency [chlordiazepoxide F(5,47)=7.62,p<0.0001; diazepamF(4,38)=15.17,p<0.0001]. The lowest effective dose of chlordiazepoxide(3.0 mg/kg, PO) decreased exit latency by 58% relative tovehicle-treated controls. Higher doses of chlordiazepoxide tested (10,30, and 100 mg/kg, PO) also significantly decreased exit latency, by97%, 72%, and 54%, respectively, relative to vehicle-treated animals.The U-shaped curve for these effects of chlordiazepoxide is probably dueto the sedative properties of this drug. The lowest effective dose ofdiazepam (1.0 mg/kg, PO) decreased exit latency by 62% relative tocontrol, and the higher doses tested (3.0 and 10 mg/kg, PO) furtherdecreased exit latency by 69% and 91%, respectively, relative tovehicle-treated controls.

(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrilewas tested at single doses (1.0, 1.8, and 3.0 mg/kg, PO) in threeseparate studies, followed by a complete dose-response (0.56-3.0 mg/kg,PO) study. In all single-dose studies, vehicle-treated animals showedlong latencies to exit the dark chamber and explore the open field. Themean exit latencies were 780±81, 748±77, and 824±76 s in the threestudies, respectively (87%, 83%, and 92%, respectively, of the totaltest duration of 900 s). The positive control DMP696 (10 mg/kg), anotherCRF₁ receptor antagonist, significantly reduced exit latency in each ofthe three studies (decrease of 57%, 74%, and 81%, respectively). Thedoses of 1.8 and 3.0 mg/kg, but not of 1.0 mg/kg, of(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethyylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrlesignificantly decreased exit latency. The dose of 1.8 mg/kg reduced exitlatency by 53% [F(2,23)=7.52, p=0.003] and the dose of 3.0 mg/kg by 51%[F(2,23)=14.05, p=0.0001].

In the dose-response study, vehicle-treated animals showed an exitlatency of 747±78 s (83% of the total test duration of 900 s). Thepositive control, DMP696 at 10 mg/kg, significantly reduced exit latency(62% decrease). Consistent with the effects shown in the single-dosestudies,(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitriledose-dependently reduced exit latency in the situational anxiety modelwhen administered orally [F(5,47)=3.69, p=0.007). The lowest effectivedose of(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrilewas 1.8 mg/kg, which resulted in a decrease in exit latency of 60%relative to vehicle-treated controls. A higher dose of(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile(3.0 mg/kg) also significantly decreased exit latency by 56%. Lowerdoses of(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile(0.56 and 1.0 mg/kg) did not significantly alter exit latency. Insummary,(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrileshowed comparable potency to the benzodiazepines diazepam andchlordiazepoxide in producing anxiolytic-like effects in the ratsituational anxiety model.

Pharmaceutical Composition and Methods of Use

Compound I demonstrates inhibition of CRF. Inhibition of CRF correlateswith efficacy for psychiatric disorders and neurological diseasesincluding depression, anxiety related disorders, irritable bowelsyndrome, addiction and negative aspects of drug and alcohol withdrawal,and other conditions associated with CRF. As such, Compound I can beuseful for the treatment of these disorders and other aspects of theinvention are compositions and methods of using Compound I to treatthese conditions and other conditions associated with aberrant levels ofCRF.

Another aspect of the invention is a method for the treatment ofpsychiatric or neurological conditions associated with CRF whichcomprises administering a therapeutically effective amount of(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrileto a patient.

Another aspect of the invention is a method for the treatment ofdepression.

Another aspect of the invention is a method for the treatment of anxietyor an anxiety related disorder.

Another aspect of the invention is a method for the treatment ofirritable bowel syndrome.

Another aspect of the invention is a method for the treatment ofaddiction or negative aspects of drug and alcohol withdrawal.

Compound I is generally given as a pharmaceutical composition comprisedof a therapeutically effective amount of Compound I, or apharmaceutically acceptable salt, and a pharmaceutically acceptablecarrier and may contain conventional excipients. A therapeuticallyeffective amount is the amount needed to provide a meaningful patientbenefit as determined by practitioners in that art. Pharmaceuticallyacceptable carriers are those conventionally known carriers havingacceptable safety profiles. Compositions encompass all common solid andliquid forms including capsules, tablets, losenges, and powders as wellas liquid suspensions, syrups, elixers, and solutions. Compositions aremade using common formulation techniques and conventional excipients(such as binding and wetting agents) and vehicles (such as water andalcohols).

Solid compositions are normally formulated in dosage units providingfrom about 1 to about 1000 mg of the active ingredient per dose. Someexamples of solid dosage units are 1 mg, 10 mg, 100 mg, 250 mg, 500 mg,and 1000 mg. Liquid compositions are generally in a unit dosage range of1-100 mg/mL. Some examples of liquid dosage units are 1 mg/mL, 10 mg/mL,25 mg/mL, 50 mg/mL, and 100 mg/mL.

The invention encompasses all conventional modes of administration; oraland parenteral methods are preferred. Typically, the daily dose will be0.01-100 mg/kg body weight daily. Generally, more compound is requiredorally and less parenterally. The specific dosing regime, however,should be determined by a physician using sound medical judgment.

Description of the Specific Embodiments

In the following examples, all temperatures are given in degreesCentigrade. Melting points were recorded on a Meltemp 3.0 LaboratoryDevices, Inc. or Thomas Scientific Unimelt capillary melting pointapparatus and are uncorrected. Proton magnetic resonance (¹H NMR)spectra were recorded on a Bruker 400 or a Bruker 500 MHz spectrometer.All spectra were determined in the solvents indicated and chemicalshifts are reported in δ units downfield from the internal standardtetramethylsilane (TMS) and interproton coupling constants are reportedin Hertz (Hz). Multiplicity patterns are designated as follows: s,singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broadpeak; dd, doublet of doublet; br d, broad doublet; dt, doublet oftriplet; br s, broad singlet; dq, doublet of quartet. Optical rotations[(α]_(D) were determined on a Rudolph Scientific Autopol IV polarimeterin the solvents indicated; concentrations are given in mg/mL. Lowresolution mass spectra (MS) and the apparent molecular (M+H⁺) or(M−H)⁺was determined on a Finnegan SSQ7000. High resolution mass spectrawere determined on a Finnegan MAT900. Liquid chromatography (LC)/massspectra were run on a Shimadzu LC coupled to a Water Micromass ZQ.

Abbreviations generally follow convention terms used in the art: “1×”for once, “2×” for twice, “3×” for thrice, “° C.” for degrees Celsius,“eq” for equivalent or equivalents, “g” for gram or grams, “mg” formilligram or milligrams, “L” for liter or liters, “mL” for milliliter ormilliliters, “μL” for microliter or microliters, “N” for normal, “M” formolar, “mmol” for millimole or millimoles, “min” for minute or minutes,“h” for hour or hours, “rt” for room temperature, “RT” for retentiontime, “atm” for atmosphere, “psi” for pounds per square inch, “conc.”for concentrate, “sat” or “sat'd ” for saturated, “MW” for molecularweight, “mp” for melting point, “ee” for enantiomeric excess, “MS” or“Mass Spec” for mass spectrometry, “ESI” for electrospray ionizationmass spectroscopy, “HR” for high resolution, “HRMS” for high resolutionmass spectrometry, “LCMS” for liquid chromatography mass spectrometry,“HPLC” for high pressure liquid chromatography, “RP HPLC” for reversephase HPLC, “TLC” or “tic” for thin layer chromatography, “NMR” fornuclear magnetic resonance spectroscopy, “¹H” for proton, “δ” for delta,“s” for singlet, “d” for doublet, “t” for triplet, “q” for quartet, “m”for multiplet, “br” for broad, “Hz” for hertz, and “α”, “β”, “R”, “S”,“E”, and “Z” are stereochemical designations familiar to one skilled inthe art.

(S)-2-Cyclopropyl-2-[(S)-1-phenylethylamino]acetic acid. A stirredsolution of cyclopropane carboxaldehyde (200.0 g, 1.42 mol) andS-(−)1-1-phenyl ethylamine (172.9 g, 1.42 mol) in methanol (2.0 L) in a3-neck 5 L round bottom flask was heated at 75° C. for 2 h. The reactionmixture was then cooled to room temperature and potassium cyanide (185.0g, 1.42 mol) was added in portions. The reaction mixture was stirredovernight at room temperature. Water (600 mL) was added followed bydropwise addition of conc. HCl (250 mL) until the reaction mixturereached pH 9-10. The mixture was then extracted with ethyl acetate (4×1L) and the combined organic layers were concentrated under reducedpressure to afford a yellow oil. Conc. HCl (3.5 L) was added to thisresidue and the mixture was heated at 95° C. overnight. After cooling toroom temperature, 10% potassium hydroxide solution was added dropwisewith cooling and stirring until the pH was nearly neutral (pH 7-8).Stirring was continued for an additional 45 min at 0° C. The contentswas filtered and washed thoroughly with cold methanol (5 L). Themoisture content at this stage was observed to be about 8-10%. The solidwas re-suspended in dry acetone (7 L) and filtered to obtain(S)-2-cyclopropyl-2-[(S)-1-phenylethylamino]acetic acid (330 g, 52%yield) as a solid with <1% moisture: ¹H NMR (400 MHz, DMSO-d₆) δ7.44-7.26 (m, 5H), 3.91-3.86 (m, 1H), 2.28-2.22 (m, 1H), 1.34 (d, J=4.0Hz, 3H), 0.93-0.89 (m, 1H), 0.41-0.37 (m, 2H), 0.31-0.27 (m, 1H),0.11-0.07 (m, 1H).

(S)-2-Cyclopropyl-2-[(S)-1-phenylethylamino]ethanol. To a solution of(S)-2-cyclopropyl-2-[(S)-1-phenylethylamino]acetic acid (400 g, 1.82mol) in dry THF (5.2 L) at 0° C. was added borane dimethylsulfide (neat)(485 g, 6.39 mol) with vigorous stirring. The reaction mixture wasstirred overnight at room temperature. Reaction progress was monitoredby HPLC. Stirring was continued until the acid was consumed completely(18-20 h). Upon completion the reaction mixture was cooled to 0° C. andmethanol (6 L) was added dropwise. The mixture was concentrated undervacuum and the residue was dissolved in chloroform (5 L). The organiclayer was washed with 10% aqueous NaHCO₃ solution (2×1 L) followed bybrine, dried over Na₂SO₄, filtered and concentrated to afford atan-yellow oil. The oil was distilled under reduced pressure to afford(S)-2-cyclopropyl-2-[(S)-1-phenylethylamino]ethanol (166 g, 44% yield)as a colorless oil: bp 175-184° C., 0.1 mm Hg; [α]²⁵ _(D)−52.1 (c 1.0,CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ ¹H NMR (400 MHz, CDCl₃) δ 7.33-7.20(m, 5H), 3.91-3.86 (m, 1H), 3.67 (dd, J_(AB)=10.6, J_(AX)=3.8 Hz, 1H),3.39 (dd, J_(BA)=10.6, J_(BX)=4.3 Hz, 1H), 2.40 (s br, 2H), 1.70-1.65(m, 1H), 1.35 (d, J=6.6 Hz, 3H), 0.88-0.82 (m, 1H), 0.47-0.37 (m, 2H),0.05-0.06 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 146,8, 127.6, 126.2,125.9, 63.0, 60.5, 54.4, 24.4, 13.3, 3.0, 1.5; LRMS (ESI) m/e 206.3[(M+H)⁺, calcd for C₁₃H₂₀NO 206.2].

(S)-1-Cyclopropyl-2-methoxy-N-[(S)-1-phenylethyl]ethanamine. To asolution of (S)-2-cyclopropyl-2-[(85-1-phenylethylamino]ethanol (29.2 g,0.143 mol) in THF (700 mL) at 0° C. was added NaH (6.29 g, 0.157 mol,60% dispersion in mineral oil). The cooling bath was removed and thereaction mixture was allowed to warm to room temperature and was stirredat room temperature for 30 min. Methyl iodide (20.30 g, 0.143 mol) wasthen added dropwise via syringe. Some warming occurred soon after theaddition was complete. The temperature of the reaction mixture wascontrolled at 25° C. with a water bath containing a small amount of ice.The reaction mixture was stirred at room temperature for 4 h. Thereaction mixture was then slowly quenched with saturated aqueous NaHCO₃solution and was transferred to a separatory funnel containing saturatedaqueous NaHCO₃ solution (400 mL). The aqueous layer was extracted withethyl acetate (3×300 mL). The combined organic layers were washed withbrine, dried over MgSO₄, filtered and concentrated. The crude productwas purified by column chromatography on silica gel (5% MeOH in 1:1ethyl acetate/hexanes) to afford(S)-1-cyclopropyl-2-methoxy-N-[(S)-1-phenylethyl]-ethanamine (26.52 g,85% yield) as a light brown oil: [α]²⁵ _(D)−61.5 (c 0.72, CHCl₃); ¹H NMR(400 MHz, CDCl₃) δ 7.30-7.25 (m, 4H), 7.20-7.16 (m, 1H), 3.98-3.93 (q,J=6.8 Hz, 1H), 3.50 (dd, J_(AB)=9.5, J_(AX)=3.5 Hz, 1H), 3.35 (s, 3H),3.34 (dd, J_(BA)=9.5,J_(BX)=5.8 Hz, 1H), 1.79-1.74 (m, 2H), 1.33 (d,J=6.5 Hz, 3H), 0.73-0.67 (m, 1H), 0.36-0.34 (m, 2W), 0.03-0.06 (m, 2H);GC/MS (ESI) m/e 220.2 [(M+H)⁺, calcd for C₁₄H₂₂NO 220.2].

(S)-1-Cyclopropyl-2-methoxyethanamine hydrochloride.(S-1-cyclopropyl-2-methoxy-N-[(S)-1-phenylethyl]ethanamine (100 g, 458mmol) was combined with Pd(OH)₂/C (50 g, 20% on carbon) and ethanol (1.2L) in a Parr bottle. The reaction mixture was placed under an H₂atmosphere (15 psi) and was shaken for 18 h. The reaction mixture wasthen filtered through a pad of Celite into a flask containing 2 N HCl inEt₂O (360 mL) with stirring. The resulting filtrate was concentrated toa yellow solid, which was then co-evaporated with Et₂O (500 mL). Theresulting solid was dried overnight in vacuo to give(S)-1-cyclopropyl-2-methoxyethanamine hydrochloride (69 g, 99% yield) asa white solid: mp 190-192° C.; [α]²⁵ _(D)+16.3 (c 0.446, MeOH); ¹H NMR(400 MHz, CDCl₃) δ 8.41 (s br, 3H), 3.68 (d, J=5.6 Hz, 2H), 3.39 (s,3H), 2.64-2.60 (m, 1H), 1.20-1.13 (m, 1H), 0.71-0.58 (m, 3H), 0.32-0.28(m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 72.1, 59.2, 57.5, 10.7, 4.2, 4.1;LRMS (ESI) m/e 231.2 [(2M+H)⁺, calcd for C₁₂H₂₇N₂O₂ 231.2].

N,2-Dimethoxy-N-methylacetamide. Triethylamine (115 mL) was added to asolution of methoxyacetic acid (35.0 g, 389 mmol) in CH₂Cl₂ (1200 mL) atroom temperature. N,O-dimethylhydroxylamine hydrochloride (45.5 g, 467mmol) was added, and after stirring for 5 min, the suspension was cooledto 0° C. Ethyl diazocarboxylate (EDC) (81.7 g, 428 mmol) was then addedand the reaction mixture was stirred overnight while allowing it to warmto room temperature. The mixture was poured into a separatory funnel anddiluted with CH₂Cl₂ (500 mL). The organic layer was washed with 1 N HCl(2×300 mL), saturated aqueous NaHCO₃ solution (2×300 mL), and brine (300mL). The resulting solution was dried over MgSO₄, filtered andconcentrated in vacuo. The product was purified by column chromatographyon a short column of silica gel (5% methanol in CH₂Cl₂) to affordN,2-dimethoxy-N-methylacetamide (33.2 g, 64% yield) as a colorless oil:¹H NMR (400 MHz, CDCl₃) δ 4.20 (s, 2H), 3.67 (s, 3H), 3.45 (s, 3H), 3.17(s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 173.0, 69.7, 69.4, 61.4, 59.4; LRMS(ESI) m/e 134.1 [(M+H)⁺, calcd for C₅H₁₂NO₃ 134.1].

1-Cyclopropyl-2-methoxyethanone. Magnesium turnings (15.2 g, 632 mmol)were added to a 5-L 3-necked flask equipped with an addition funnel. Theflask, funnel, and turnings were flame dried and then a reflux condenserwas then placed on the flask. After the flask and contents had cooled toroom temperature, diethyl ether (100 mL) was added to the flask,followed by addition of a portion of cyclopropyl bromide (5.0 mL, 7.55g, 62.4 mmol) and several crystals of iodine. After the reaction hadinitiated, diethyl ether (400 mL) was added to the reaction flask. Theremaining cyclopropyl bromide (87.3 g, 721 mmol) was then added slowlyover 30 min with intermittent cooling of the reaction mixture with anice-water bath. After the addition was complete and the magnesium wasconsumed, additional diethyl ether (700 mL) was added and the reactionmixture was cooled to 0° C. The magnetic stirrer was replaced with amechanical stirrer and a solution of N,2-dimethoxy-N-methylacetamide(42.07 g, 316 mmol) dissolved in diethyl ether (500 mL) was added slowlyover 30 min via the addition funnel. A white solid formed during thistime. After the addition was complete, the cooling bath was removed andthe mixture was stirred at room temperature for 1 h. The mixture wasthen cooled to 0° C. and the reaction was quenched by the addition of 1N HCl (700 mL, added slowly at first). After stirring for an additional15 min, the mixture was transferred to a separatory funnel, and theaqueous layer was extracted with ether (3×500 mL). The combined organiclayers were washed with saturated aqueous NaHCO₃ solution (400 mL),brine (400 mL), dried over MgSO₄, filtered and concentrated with minimalvacuum (500 mbar). The product was purified by distillation underreduced pressure while submerging the collection flask in a dryice/isopropanol bath to afford 1-cyclopropyl-2-methoxyethanone (29.2 g,81% yield) as a colorless oil: bp 35-38° C., 5 mm Hg; ¹H NMR (400 MHz,CDCl₃) δ 4.13 (s, 2H), 3.43 (s, 3H), 2.11-2.07 (m, 1H), 1.10-1.06 (m,2H), 0.94-0.89 (m, 2f); GC/MS (CI) m/e 115.1 [(M+H)⁺, calcd for C₆H₁₁O₂115.1].

Benzyl 1-cyclopropyl-2-methoxyethylcarbamate.1-Cyclopropyl-2-methoxyethanone (10.0 g, 88.0 mmol3 in THF (1000 mL) wastreated with ammonium trifluoroacetate (115 g, 880 mmol) and the mixturewas cooled to 0° C. Sodium triacetoxyborohydride (27.9 g, 133 mmol) wasadded, the cooling bath was removed and the reaction mixture was gentlyheated at 40° C. with a warm water bath for 2 h. The mixture was cooledto room temperature and concentrated to give1-cyclopropyl-2-methoxyethanamine which was used directly in the nextstep.

Crude 1-cyclopropyl-2-methoxyethanamine from the previous step wasdissolved in CH₂Cl₂/H₂O (300 mL/300 mL) and Na₂CO₃ (111.9 g, 1.06 mol)was added. The reaction mixture was placed in an ice bath and CbzCl(16.46 g, 96.78 mmol) was added via syringe. During the addition, theinternal reaction mixture temperature was maintained at 15-20° C. Afterthe addition was complete, the reaction mixture was stirred at roomtemperature for 2 h. The mixture was poured into a separatory funnel,diluted with H₂O, (300 mL), and extracted with CH₂Cl₂ (3×300 mL). Thecombined organic layers were washed with brine (300 mL), dried overMgSO₄, filtered and concentrated. The product was purified by columnchromatography on silica gel (30% ethyl acetate in hexanes) to fairnishbenzyl 1-cyclopropyl-2-methoxyethylcarbamate (17.2 g, 78% yield for 2steps) as an oil which crystallized upon standing: mp 190.5-192° C.; ¹HNMR (400 MHz, DMSO-d₆) δ 7.39-7.29 (m, 5H), 7.17 (d, J=8.5 Hz, 1H), 5.00(s, 2H), 3.36-3.34 (m, 2H), 3.23 (s, 3H), 3.19-3.14 (m, 1H), 0.85-0.79(m, 1H), 0.43-0.37 (m, 1H), 0.35-0.22 (m, 2H), 0.20-0.16 (m, 1H), LRMS(ESI) m/e 250.3 [(M+H)⁺, calcd for C₁₄H₂₀NO₃ 250.1].

(S)-Benzyl 1-cyclopropyl-2-methoxyethylcarbamate. Racemic1-cyclopropyl-2-methoxyethylcarbamate was separated into its enantiomersby HPLC: Chiralpak AD column (10 cm×50 cm), 94% heptane/6% ethanol, 300mL/min, λ=210 nm, 1 gram per injection, 30 min method, Peak 1 (S), Peak2 (R) and was determined to have an optical purity>99% ee by analyticalHPLC (Chiralpak AD column, 4.6×250 mm, 95% heptane/5% ethanol, 0.8mL/min, λ=212 nm, t_(R)=15.79 min): mp 190.5-192° C.; [α]²⁵ _(D)−18.2 (c0.500, CHCl₃); ¹H NMR (400 MHz, DMSO-d₆) δ 7.39-7.29 (m, 5H), 7.17 (d,J=8.5 Hz, 1H), 5.00 (s, 2H), 3.36-3.34 (m, 2H), 3.23 (s, 3H), 3.19-3.14(m, 1H), 0.85-0.79 (m, 1H), 0.43-0.37 (m, 1H), 0.35-0.22 (m, 2H),0.20-0.16 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 155.6, 136.9, 128.0,127.38, 127.34, 74.0, 64.8, 57.8, 53.5, 12.6, 2.2, 1.5; LRMS (ES⁺) m/e272.3 [(M+Na)⁺, calcd for C₁₄H₁₉NO₃Na 272.1].

(S)-1-cyclopropyl-2-methoxyethanamine hydrochloride. To a solution of(S)-1-cyclopropyl-2-methoxyethylcarbamate (4.36 g, 17.5 mmol) in EtOH(80 ml) and CHCl₃ (3 mL) in a Parr bottle was added 4 N HCl in dioxane(5 mL) and Pd/C (476 mg, 10%, wet, Degussa type). The mixture was placedon a Parr shaker under H₂ atm at 45 psi for 16 h. The reaction mixturewas filtered through a pad of Celite and the filtrate was concentratedthen reconcentrated from hexanes (2×) to afford(S)-1-cyclopropyl-2-methoxyethanamine hydrochloride (2.65 g, 100% yield)as a white solid: mp 190-192° C.; [α]²⁵ _(D)+14.3 (c 0.446, MeOH); ¹HNMR (400 MHz, CDCl₃) δ 8.41 (s br, 3H), 3.68 (d, J=5.6 Hz, 2H), 3.39 (s,3H), 2.64-2.60 (m, 1H), 1.20-1.13 (m, 1H), 0.71-0.58 (m, 3H), 0.32-0.28(m, 1H); ³C NMR (100 MHz, CDCl₃) δ 72.1, 59.2, 57.5, 10.7, 4.2, 4.1;LRMS (ESI m/e 231.2 [(2M+H)⁺, calcd for C₁₂H₂₇N₂O₂ 231.2].

(S)-2-(1-Cyclopropyl-2-methoxyethylamino)acetonitrile hydrochloride.Chloroacetonitrile (8.40 mL, 133 mmol) was added to a stirred suspensionof (S)-1-cyclopropyl-2-methoxyethanamine hydrochloride (20.0 g, 133mmol), K₂CO₃ (52.0 g, 376 mmol) and KI (24.2 g, 145 mmol) inacetonitrile (300 mL) at room temperature. The mixture was stirred at48° C. for 17 hours. The reaction mixture was then cooled to roomtemperature then filtered through a pad of Celite. The resultingfiltrate was concentrated to a dark brown semi-solid. The solid wassuspended in CH₂Cl₂ and purified by column chromatography on silica gel(CH₂Cl₂→3% methanol in CH₂Cl₂) to yield a brown oil (18.8 g). The oilwas dissolved in Et₂O (150 mL) and the solution was acidified with 2 NHCl in Et₂O (100 mL) to give(S)-2-(1-cyclopropyl-2-methoxyethylamino)-acetonitrile hydrochloride(23.6 g, 93% yield) as an off-white solid: [α]²⁵ _(D)+22.9 (c 0.714,CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 10.51 (s br, 2H), 4.39 (ABq,J_(AB)=16.7, Δν24.4 Hz, 2H), 3,94 (dd, J_(AB)=10.6, J_(AX)=7.8 Hz, 1H),3.79 (dd, J_(BA)=10.8, J_(BX)=2.2 Hz, 1H), 3.42 (s, 3H), 2.79-2.75 (m,1H), 1.25-1.17 (m, 1H), 0.90-0.83 (m, 1H), 0.78-0.70 (m, 2H), 0.39-0.34(m, 1H); LRMS (ES⁺) m/e 155.2 [(M+H)⁺, calcd for C₈H₁₅N₂O 155.1].

(S)-3,5-Dibromo-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one.(S)-2-(1-cyclopropyl-2-methoxyethylamino)acetonitrile hydrochloride(15.0 g, 78.7 mmol) was suspended in anhydrous dichloromethane (300 mL)in a 1 L, 3-necked round bottom flask equipped with an addition funnel.The mixture was cooled to −60° C. and oxalyl bromide (41.3 mL, 440 mmol)was added dropwise over 15 min. After addition was complete, the coolingbath was removed and the reaction mixture was allowed to warm to roomtemperature and was then heated at 40° C. for 3 hours. The mixture wascooled to room temperature and concentrated under vacuum. The solid waspurified by direct addition to the head of a silica gel column andeluted (5%→20% ethyl acetate in hexanes) after residual oxalyl bromidehad stopped reacting with the silica gel to give(S)-3,5-dibromo-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one (15.5g, 56% yield) as an off-white solid: np 98.5-100.5° C.; [α]²⁵D−71.8 (c1.19, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.61 (s, 1H), 4.08-4.04 (m, 1H),3.72 (dd, J_(AB)=10.5, J_(AX)=45 Hz, 1H), 3.61 (dd, J_(BA)=10.3,J_(BX)=3.0 Hz, 1H), 3.32 (s, 3H), 1.41-1.36 (m, 1H), 0.82-0.76 (m, 1H),0.65-0.59 (m, 1H), 0.54-0.48 (m, 1H), 0.32-0.27 (m, 1H); LRMS (APCI) m/e351.1 [(M+H)⁺, calcd for C₁₀H₁₃N₂O₂Br₂ 350.9].

(S)-3,5-Dichloro-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one.Oxalyl chloride (54.5 mL, 624 mmol) was added dropwise to a cold (<8°C.) solution of (S)-2-(1-cyclopropyl-2-methoxyethylamino)acetonitrilehydrochloride (23.6 g, 124 mmol) in 1,4 dioxane (300 mL) and methylenechloride (200 mL). The reaction mixture was then heated at 53° C. for 19h. The reaction mixture was cooled to room temperature and concentratedto a semi-solid then co-evaporated three times with CH₂Cl₂ (50 mL). Theresulting brown solid was purified by column chromatography on silicagel (0→10%→20% ethyl acetate in hexanies) to afford(S)-3,5-dichloro-1-(1-cyclopropyl-2-methoxyethylpyrazin-2(1H)-one (22.4g, 69% yield) as a white solid: Mp 109.8-110.8° C.; [α]²⁵ _(D)−88.8 (c0.513, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.54 (s, 1H), 4.12-4.08 (m,1H), 3.73 (dd, J_(AB)=10.3, J_(AX)=4.5 Hz, 1H), 3.62 (dd, J_(BA)=10.3,J_(BX)=3.0 Hz, 1H), 3.32 (s, 3H), 1.43-1.37 (m, 1H), 0.82-0.76 (m, 1H),0.65-0.61 (m, 1H), 0.55-0.50 (m, 1H), 0.33-0.27 (m, 1H); LRMS (ESI) m/e206.3 [(M+H)⁺, calcd for C₁₀H₁₃N₂O₂Cl₂ 263.0]. Anal. calcd. forC₁₀H₁₂N₂O₂Cl₂; C, 45.64, H, 4.59, N, 10.64. Found: C, 45.74, H, 4.62, N,10.61.

(S)-3,5-dichloro-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one [160g, 90-95% of the (S)-enantiomer] from various batches was processed byThar SFC to isolate the (S)-enantiomer in >99% enantiomeric purity. Atotal of 133 g of(S)-3,5-dichloro-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one wasisolated in 3 batches. 11.8 g of the undesired isomer was also obtained.The enantiomeric excess (ee) of the compounds delivered were >99%.Preparative conditions on Thar 350 SFC were as follows: amount racemate(g): 65, 58, 37; column: Chiralpak AD-H, 5×25 cm; mobile phase: 10% EtOHin CO₂; pressure (bar): 100; flow rate (ml/min): 200; solutionconcentration (mg/ml): 50; injection amount (ml): 6; Cycle time(min/inj): 3.5; temperature: 35; throughput (g/hr): 5.1; Detector λ:220.

3-Methyl-5-nitropyridin-2-ol. A 3-necked, 2-L, round-bottomed flaskequipped with a mechanical stirrer, an addition funnel and a thermometerwas placed in an ice-water bath. Conc. H₂SO₄ (150 mL) was added to theflask. 2-Amino-3-methylpyridine (50.0 g, 0.463 mol, Lancaster, CAS1603-40-3, mp. 29° C., prewarmed in a warm water bath to melt it) wasweighed out in a 125 mL Erlenmeyer flask and was subsequently added insmall portions via a prewarmed Pasteur pipet with the narrow tip brokenoff. The Erlenmeyer flask was kept in a warm water bath during theaddition to prevent the starting material from solidifying. Thetemperature rose to ca. 45° C. during the addition and white smoke/fogformed within the flask. Conc. H₂SO₄ (100 mL) was added to the residualstarting material and the mixture was added to the reaction flask. Theresulting mixture was a milky-white suspension. A solution of conc.H₂SO₄ (35 mL) and 70% nitric acid (35 mL) was premixed with ice-waterbath cooling and transferred into the addition funnel. After theinternal temperature of the reaction mixture had cooled to 10-15° C.(but not below 10° C.), the premixed H₂SO₄/HNO₃ acid mixture was addeddropwise at a rate such that the internal reaction temperature rose to20-25° C. (5-10 min addition time). After the addition was complete, theice-water bath was replaced with a tap-water bath. The reactiontemperature slowly increased to ca. 30° C. range and then cooled down toroom temperature. The reaction should be monitored during this time toensure that the temperature does not rise too high. The reaction mixturewas then stirred overnight and then 70% nitric acid (35 mL) was addeddropwise via the addition funnel to the dark red-brown mixture at a rateof addition such that the temperature did not exceed 35° C. At thistime, the reaction flask was sitting in a water bath containing water atroom temperature. Water (500 mL) was then added to the reaction flask inportions via addition funnel. The first ca. 150 mL of water was addeddropwise while allowing the internal temperature to climb slowly to50-60° C. The rate of stirring was increased in order to break up anyfoaming that occurred. Brown gas evolved during the addition of theinitial ca. 150 mL of water. The remaining ca. 350 mL of water was addedat a faster rate after gas evolution had stopped and a temperatureincrease was no longer observed. The reaction turned from a dark cloudybrown to a clear orange solution. Some yellow precipitate may form asthe reaction cools to below 50° C. The water bath was then removed, andreplaced with a heating mantle, and the addition funnel was replacedwith a condenser. The reaction mixture (a light orange solution orbright yellow solution) was then heated at 115-118° C. for 1.75-2 h.Additional gas evolution occurred at ca. 115° C. during this time. Thereaction mixture was then cooled to room temperature with the aid of anice-water bath and was then cooled further to 0° C. by adding icedirectly into the reaction mixture. The solid that formed was collectedon a Buchner funnel and was washed with cold water followed by a minimalamount of cold ethanol followed by a minimal amount of cold ether. Thesolid was then dried under vacuum to afford 3-methyl-5-nitropyridin-2-ol(53.5 g, 75% yield) as a pale yellow solid. mp 228-229° C.; ¹H NMR (400MHz, DMSO-d₆) δ 12.55 (s, br, 1H), 8.54 (d, J=3.0 Hz, 1H), 8.04 (d,J=2.0 Hz, 1H), 2.04 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 162.2, 135.4,130.0, 129.5, 128.1, 15.8; LRMS, (ESI) m/e 152.96 [(M−H)⁻, calcd forC₆H₅N₂O₃, 153.03]. Anal. calcd. for C₆H₆N₂O₃; C, 46.75, H, 3.92, N,18.17. Found: C, 46.80, H, 3.79, N, 18.14.

2-Methoxy-3-methyl-5-nitropyridine. 3-Methyl-5-nitropyridin-2-ol (134 g,0.872 mol) (J. Org. Chem. 1949, 14, 328-332) was divided into 3 portionsand placed in three 1-L round bottom flasks. POCl₃ (200 mL) was added toeach flask and the mixtures were heated to reflux for 2 h. The solutionswere cooled and the excess POCl₃ was removed in vacuo. The residues werepoured into ice water (1 L) with stirring and the precipitates werecollected by filtration and air dried for 20 min. The combined productswere recrystallized from 10% ethyl acetate in hexanes (300 mL) and airdried to give 2-chloro-3-methyl-5-nitropyridine (139 g, 92% yield) as awhite solid which was used in the next step without furtherpurification: ¹H NMR (300 MHz, CDCl₃) δ 9.04 (d, J=2.5 Hz, 1H), 8.33 (d,J=2.2 Hz, 1H), 2.50 (s, 3H).

2-Chloro-3-methyl-5-nitropyridine (139 g, 0.806 mol) from the aboveprocedure was divided into two portions and placed in two 2-L roundbottom flasks with methanol (500 mL). The solutions were cooled in dryice/isopropanol baths as solid sodium methoxide (26.5 g, 0.467 mol) wasadded portion-wise to each flask so that the temperature was remainedbelow 20° C. When the additions were complete, the resulting mixtureswere heated to reflux for 1 h. The mixtures were cooled and diluted withice water (500 mL) to give white precipitates, which were collected byfiltration. The combined filtrates were washed with water and air driedto give 2-methoxy-3-methyl-5-nitropyridine (127 g, 97% yield) as a whitesolid: ¹H NMR (400 MHz, CDCl₃) δ 8.91 (d, J=2.0 Hz, 1H), 8.16 (s, 1H),4.06 (s, 3H), 2.25 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 165.83, 141.91,139.37, 132.92, 121.77, 54.83, 15.84. An analytical sample wasrecrystallized from hexane to give white needles, mp 95-96.5° C. Anal.calcd. for C₇H₈N₂O₃: C, 50.00, H, 4.79, N, 16.66. Found: C, 49.73, H,5.02, N, 16.48.

tert-Butyl 2-(6-methoxy-5-methyl-3-nitropyridin-2-y)acetate. A yellowsolution of 2-methoxy-3-methyl-5-nitropyridine (68.8 g, 409 mmol) andtert-butyl 2-chloroacetate (77.0 g, 511 mmol) in THF (1 L) was stirredand cooled to −20° C. in a dry ice/isopropanol bath. Potassiumtert-butoxide (115 g, 1.02 mol) was added at a rate so that the reactiontemperature was less than −10° C. The reaction mixture turned darkpurple. When the addition was complete, the cooling bath was removed andthe reaction was stirred for 30 min. The stirred reaction mixture wasquenched with HCl (500 mL, 2.4 N). The purple solution turned paleyellow and the mixture separated into two layers. The organic layer wasseparated, washed three times with brine, and concentrated in vacuo.Hexane was added to the amber residue. The mixture was concentrated invacuo and then dried under high vacuum for 1 hr to give tert-butyl2-(6-methoxy-5-methyl-3-nitropyridin-2-yl)acetate (83.4 g, 72% yield) asa tan solid: ¹H NMR (400 MHz, CDCl₃) δ 8.16 (s, 1H), 4.09 (s, 2H), 4.02(s, 2H), 2.21 (s, 3H), 1.44 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 168.81,163.63, 147.88, 139.41, 135.19, 120.82, 81.66, 54.69, 44.48, 28.03,15.27. An analytical sample was recrystallized from hexane to give whiteneedles, mp 71-72.5° C. Anal. calcd. for C₁₃H₁₈N₂O₅: C, 55.31, H, 6.42,N, 9.92. Found: C, 55.52, H, 6.40, N, 9.84.

2-(6-Methoxy-5-methyl-3-nitropyridin-2-yl)acetic acid. A solution oftert-butyl 2-(6-methoxy-5-methyl-3-nitropyridin-2-yl)acetate (83.0 g,294 mmol) in TFA (200 mL) was heated in a hot water bath for 1 h. Thesolution was concentrated in vacuo to give a brown oil. The oil wasdiluted with hexane and stirred. The resulting solid was collected byfiltration and air dried to give2-(6-methoxy-5-methyl-3-nitropyridin-2-yl)acetic acid (62.8 g, 94%yield) as a tan solid: ¹H NMR (400 MHz, CDCl₃) δ 8.70 (s br, 1H), 8,20(s, 1H), 4.25 (s, 2H), 4.03 (s, 3H), 2.24 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 175.59, 163.80, 146.52, 139.16, 135.30, 121.53, 54.86, 42.88,15.32. An analytical sample was recrystallized from hexane: mp 135-137°C. Anal. calcd. for C₉H₁₀N₂O₅: C, 47.79, H, 4.46, N, 12.39. Found: C,47.65, H, 4.14, N, 12.30.

2-Methoxy-3,6-dimethyl-5-nitropyridine. A mixture of tert-butyl2-(6-methoxy-5-methyl-3-nitropyridin-2-yl)acetate (62.5 g, 276 mmol),K₂CO₃ (20.0 g, 145 mmol), and DMF (100 mL) was heated with stirring in ahot water bath to 90° C. for 1 h. Gas evolution was noted during theheating period and had ceased after 1 hour. The mixture was poured intostirred ice water (600 mL), with washing of the reaction flask with asmall volume of acetone. The resulting precipitate was collected byfiltration and air dried to give 2-methoxy-3,6-dimethyl-5-nitropyridine(48.5 g, 96% yield) as a tan solid: ¹H NMR (400 MHz, CDCl₃) δ 8.08 (S,1H), 4.02 (s, 3H), 2.76 (d, 3H), 2.19 (s, 3H); ¹³C NMR (100 MHz, CDCl₃)δ 163.37, 151.63, 139.44, 135.02, 119.39, 54.45, 24.18, 15.16. Ananalytical sample was recrystallized from hexanes to give tan needles:mp 85.9-90.5° C. Anal. calcd. for C₈H₁₀N₂O₃: C, 52.74, H, 5.53, N,15.37. Found: C, 52.82, H, 5.28, N, 15.45.

2-Methoxy-3,6-dimethyl-5-nitropyridine. Dimethyl sulfoxide (35 mL) wasadded to a dry round-bottomed flask containing NaH (1.82 g, 45.5 mmol,60% in mineral oil). The resulting suspension was heated at 70° C. for35 min during which time the suspension became a solution. The reactionmixture was cooled to room temperature, trimethylsulfoxonium iodide(10.0 g, 45.5 mmol) was added, and the mixture was stirred at roomtemperature for 30 min. 2-Methoxy-3-methyl-5-nitropyridine (4.50 g,26.80 mmol) was added and the resulting dark red solution was stirred atroom temperature for 30 min, at which time TLC showed completeconsumption of starting material. The reaction mixture was transferredto a separatory funnel containing water (30 mL), and the aqueous layerwas extracted with EtOAc (3×100 mL). The combined organic layers werewashed with brine, dried over MgSO₄, filtered and concentrated. Theresidue was purified by MPLC (silica gel, 20% ethyl acetate in hexanes)to afford 2-methoxy-3,6-dimethyl-5-nitropyridine (2.00 g, 41% yield) asa colorless solid identical to that prepared by the previous method: mp85.5-86.2° C.; ¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 4.02 (s, 3H),2.77 (s, 3H), 2.20 (s, 3H).

3,6-Dimethyl-5-nitropyridin-2-ol. A solution of2-methoxy-3,6-dimethyl-5-nitropyridine (32.3 g, 182 mmol) in 12 Nhydrochloric acid (300 mL) was heated at 100° C. for 1 h. Analysis byTLC indicated that some starting material remained, so the reaction washeated at 110° C. for an additional 45 min. The reaction mixture wascooled to room temperature and poured onto ice (400 g). When the ice hadmelted and the temperature of the resulting thick brown suspension wasstill less than 0° C., the mixture was filtered. The solid cake waswashed with water (100 mL) and allowed to dry on the filter for 30 min.The solid was then resuspended in cold (−10° C.) ethanol (150 mL),filtered, washed with cold ethanol (50 mL), and air-dried on the filterfor 1 h to afford 3,6-dimethyl-5-nitropyridin-2-ol (28.0 g, 94% yield)as a tall powder: mp 263° C.; ¹H NMR (400 MHz, DMSO-d) δ 12.42 (s br,1H), 8.03 (s, 1H), 2.61 (s, 3H), 2.01 (s, 3H); LRMS (ESI) m/e 169.3[(M+H)⁺, calcd for C₇H₉N₂O₃ 169.1]. Anal. calcd. for C₇H₈N₂O₃: C, 50.00,H, 4.79, N, 16.66. Found: C, 50.01, H, 4.59, N, 16.75.

2-(Difluoromethoxy)-3,6-dimethyl-5-nitropyridine. Sodium hydride (6.63g, 166 mmol, 60% in mineral oil) was washed with hexanes (100 mL) toremove the mineral oil and was then suspended in dry acetonitrile (1500mL) at room temperature. 3,6-Dimethyl-5-nitropyridin-2-ol (27.9 g, 166mmol) was added in portions over 30 minutes to give a yellow suspension.During the addition there was some bubbling but negligible temperaturechange. Cesium fluoride (2.50 g, 16.6 mmol) was then added followed bythe slow addition of trimethylsilyl 2-(fluorosulfonyl)difluoroacetate(36.0 mL, 182 mmol) over 30 minutes. During the addition there was somebubbling, the temperature rose from 23° C. to 30° C., and the suspensionbecame noticeably less turbid. After stirring for 15 min, TLC indicatedthat starting material still remained, so additional trimethylsilyl2-(fluorosulfonyl)difluoroacetate (6.5 mL, 33 mmol) was added over 10minutes. After an additional 15 min, TLC indicated consumption ofstarting material. The reaction was quenched by the addition of water(20 mL) dropwise at such a rate that the bubbling did not become toovigorous. After bubbling ceased, additional water (200 mL) was added.Most of the solvent was removed in vacuo and the aqueous residue wasextracted with ethyl acetate (3×200 mL). The combined organic layerswere dried over Na₂SO₄ and evaporated to a brown syrup which solidifiedupon standing. This residue was dissolved in ethanol (400 mL) anddecolorizing charcoal (15 g) was added. The suspension was heated at 70°C. for 20 min and then filtered through a pad of Celite and sand. Thefiltrate was collected and the solvent was evaporated. The residue wasdissolved in methylene chloride and the solution was evaporated to give2-(difluoromethoxy)-3,6-dimethyl-5-nitropyridine (33.4 g, 92% yield) asa pale yellow solid: mp 51-52° C.; ¹H NMR (400 MHz, CDCl₃) δ8.21 (s,1H), 7.55 (t, J=72.0 Hz, 1H), 2.76 (s, 3H), 2.30 (s, 3H). Anal. calcd.for C₈H₈N₂O₃F₂: C, 44.04, H, 3.69, N, 12.84. Found: C, 43.78, H, 3.55,N, 12.58.

Alternate route. To a suspension of 3,6-methyl-5-nitropyridin-2-ol (700mg, 4.17 mmol) in acetonitrile (70 mL) was added NaH (450 mg, 11.3 mmol,60% in mineral oil). After stirring at room temperature for 15 min,2,2-difluoro-2-(fluorosulfonyl)acetic acid (0.73 mL, 7.09 mmol) wasadded dropwise over several minutes. Some bubbling occurred during theaddition. After stirring the reaction mixture at room temperature for 15min, the reaction was quenched by the slow addition of water (10 mL).The acetonitrile was removed in vacuo and the residue was transferred toa separatory funnel containing water (50 mL). The aqueous layer wasextracted with EtOAc (3×50 mL). The combined organic layers were washedwith brine, dried over MgSO₄, filtered and concentrated. The residue waspurified by MPLC on silica gel (10% ethyl acetate in hexanes) to afford2-(difluoromethoxy)-3,6-dimethyl-5-nitropyridine (870 mg, 96% yield) asa colorless solid identical to that prepared by Method A: mp 48-49° C.;¹H NMR (400 MHz, CDCl₃) δ 8.21 (s, 1H), 7.54 (t, J=72.4 Hz, 1H), 2.76(s, 3H), 2.30 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 158.1, 151.0, 142.0,137.0, 120.0, 113.9 (t, J=255.8 Hz), 23.5, 14.7.

6-(Difluoromethoxy)-2,5-dimethylpyridin-3-amine. To a solution of2-(difluoromethoxy)-3,6-dimethyl-5-nitropyridine (33.4 g, 153 mmol) inmethylene chloride (100 mL) and ethanol (600 mL) was added 10% palladiumon charcoal (3.3 g). The resulting suspension was hydrogenated on a Parrdevice at 40 psi 12 for 1 h. TLC was used to monitor the reaction.Additional 3.3 g of palladium on charcoal were added hourly until nostarting material remained. A total of 13.2 g of Pd/C was added. Thereaction mixture was kept under an H₂ atmosphere for 2 h after the lastaddition of catalyst. The reaction mixture was filtered through Celiteand sand and the collected solids were washed with ethyl acetate (2×100mL). The filtrate was concentrated in vacuo to give a grey oil, whichwas purified by column chromatography on silica gel (35%→50% ethylacetate in hexalles) to funiish6-(difluoromethoxy)-2,5-dirnethylpyridin-3-amine (25.7 g, 89% yield) asa pale yellow oil which solidified upon cooling in a refrigerator. Theproduct was recrystallized from hexanes below 0° C. to afford whiteneedles: mp 40-42° C.; ¹H NMR (400 MHz, CDCl₃) δ 7.35 (t, J=74.0 Hz,1H), 6.84 (s, 1H), 2.27 (s, 3H), 2.15 (s, 3H); LRMS (ESI) m/e 189.2[(M+H)⁺, calcd for C₈H₁₁N₂OF₂ 189.1]. Anal calcd. for C₈H₁₀N₂OF₂: C,51.12, H, 5.38, N, 14.86. Found: C, 51.17, H, 5.29, N, 14.87.

Alternate route. To a solution of2-(difluoromethoxy)-3,6-dimethyl-5-nitropyridine (1.00 g, 4.58 mmol) inEtOH (40 mL) and acetic acid (4 mL) was added iron powder (1.26 g, 22.9mmol). The reaction mixture was heated at a vigorous reflux with the aidof a heating mantle in a flask without a stir bar. After 1.75 h, thereaction mixture was cooled to room temperature and the iron powder wasremoved by filtration through a pad of Celite. The filtrate wasconcentrated and the residue was transferred to a separatory funnelcontaining saturated aqueous NaHCO₃ (50 ml). The aqueous layer wasextracted with EtOAc (3×100 mL). The combined organic layers were washedwith brine, dried over MgSO₄, filtered and concentrated. The residue waspurified by MPLC on silica gel (20%→50% ethyl acetate in hexalnes) toafford 6-(difluoromethoxy)-2,5-dimetlhylpyridin-3-amine (774 mg, 90%yield) as a pale yellow solid identical to that prepared by Method A: mp38.4-39.4° C.; ¹H NMR (400 MHz, CDCl₃) δ 7.35 (t, J=74.5 Hz, 1H), 6.82(s, 1H), 2.26 (s, 3H), 2.14 (s, 3H); ¹³C NMR 100 MHz, CDCl₃) δ 149.6,137.4, 137.1, 127.4, 119.0, 115.0 (J=251.3 Hz), 19.3, 14.8; LRMS (ESI)m/e 189.0 [(M+H)⁺, calcd for C₈H₁₁N₂OF₂ 189.1.]

(S)-5-Bromo-1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluoromethoxy)-2,5-dimethyl-pyridin-3-ylamino]pyrazin-2(1H)-one.(S)-3,5-Dibromo-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one (18.1g, 51.7 mmol) and 6-(difluoromethoxy)-2,5-dimethylpyridin-3-amine (9.70mg, 51.7 mmol) were combined in a 2 L, 3-necked round bottom flaskequipped with a thermometer and an addition funnel and placed under N₂.THF (360 mL) was added and the mixture was cooled to 0° C. NaHMDS (109mL, 109 mmol, 1 M in THF) was added dropwise via the addition funnelover 15 min (the internal temperature was maintained below 5° C.). Afterthe addition was complete, the reaction mixture was stirred at 0° C. foran additional 30 min. The reaction was quenched by the addition ofsaturated aqueous NH₄Cl (120 mL). The mixture was transferred to aseparatory funnel containing water (100 mL) and the aqueous layer wasextracted with ethyl acetate (3×250 mL). The combined organic layerswere washed with brine, dried over MgSO₄, filtered and concentrated toafford(S)-5-bromo-1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]pyrazin-2(1H)-one(22.4 g, 95% yield) as a brown solid which was used in the next stepwithout further purification: ¹H NMR (400 MHz, CDCl₃) δ 8.40 (s, 1H),7.98 (s, 1H), 7.53 (s, 1H), 7.46 (t, J=73.7 Hz, 1H), 4.16-4.12 (m, 1H),3.74 (dd, J_(AB)=10.5, J_(AX)=5.2 Hz, 1H), 3.66 (dd, J_(BA)=10.3,J_(BX)=3.3 Hz, 1H), 3.35 (s, 3H), 2.44 (s, 3H), 2.27 (s, 3H), 1.39-1.34(m, 1H), 0.85-0.79 (m, 1H), 0.66-0.62 (m, 1H), 0.56-0.51 (m, 1H),0.34-0.29 (m, 1H); HRMS (ESI) m/e 459.0864 [(M+H)⁺, calcd forC₁₈H₂₂N₄O₃BrF₂ 459.0843].

(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile.(S)-5-Bromo-1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluoromethoxy)-2,5-dimethyl-pyridin-3-ylamino]pyrazin-2(1H)-one(22.4 g, 48.8 mmol) was dissolved in anhydrous dimethylformamide (480mL) and water (24 mL) at room temperature with magnetic stirring.Nitrogen was bubbled through the reaction mixture for 15 minutes. Zinccyanide (6.00 g, 51.0 mmol), Pd₂(dba)₃ (2.23 g, 2.40 mmol) and dppf(3.24 g, 5.85 mmol) were added and the reaction mixture was heated at120° C. for 3 h. The reaction was cooled and filtered through a pad ofCelite. The DMF filtrate was evaporated in vacuo (bath temperature <40°C.) and the residue was transferred to a separatory funnel containingsaturated aqueous NH₄Cl solution (200 mL). The aqueous layer wasextracted with EtOAc (3×400 mL). The combined organic layers were washedwith brine (300 mL), dried over MgSO₄, filtered and concentrated.Purification by column chromatography on silica gel (70%→100% CH₂Cl₂ inhexanes then 2%→5% ethyl acetate in CH₂Cl₂) afforded(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-[6-(difuoromethoxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile(14.2 g, 72% yield) as a pale yellow solid. Recrystallization from amixture of EtOH:2-BuOH (10:1) afforded pale yellow needles: mp146.8-147.7° C.; [α]²⁵D−63.7 (c 0.486, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ8.40 (s, 1H), 7.98 (s, 1H), 7.53 (s, 1H), 7.46 (t, J=73.5 Hz, 1H),4.16-4.12 (m, 1H), 3.74 (dd, J_(AB)=10.3, J_(AX)=5.0 Hz, 1H), 3.67 (dd,J_(BA)=10.4, J_(BX)=3.0 Hz, 1H), 3,35 (s, 3H), 2.44 (s, 3H), 2.27 (s,3H), 1.39-1.33 (m, 1H), 0.85-0.79 (m, 1H), 0.67-0.61 (m, 1H), 0.56-0.50(m, 1H), 0.34-0.29 (m, 1H); HRMS (ESI) m/e 406.1704 [(M+H)⁺, calcd forC₁₉H₂₂N₅O₃F₂ 406.1691]. Anal. Calcd for C₁₉H₂₁N₅O₃F₂, C, 56.29; H, 5.22;N, 17.27. Found: C, 56.38; H, 5.31; N, 17.34.

(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile(97 g, from multiple runs) enhanced in the first eluting isomer (˜97%enantiomeric purity) was combined for removal of the undesired secondeluting isomer by super critical fluid chromatography (SFC) on chiralsupport: Chiralpak OD-H column (5×25 cm), mobile phase=15%isopropanol/acetonitrile (1:1) in CO₂; flow rate=200 mL/min,pressure=100 bar, temperature=35 C, λ=254 nm, 5 mL of 91 mg/mL inisopropanol/acetonitrile (1:1) per injection per 6 min. The purifiedmaterial had an optical purity>99% ee as deteimined by analytical SFC:Chiralcel OD-H column (4.6×250 mm, 5 μm); mobile phase=8% ethanol inCO₂; flow rate=2 mL/min @ 150 bars; λ=215 nm; t_(R)=6.5 min.

The separation provided 85 g of enantiomerically pure compound (ee>99%).This material was divided into 3 portions of 28 grams each. Each portionwas recrystallized from anhydrous 2-butanol (280 mL). The combinedsolids were dried under high vacuum for 72 hours to provide 81.7 gramsof(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile.

(S)-5-Chloro-1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluoromethoxy)-2,5-dimethyl-pyridin-3-ylamino]pyrazin-2(1H)-one.(S)-3,5-Dichloro-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one (15,0g, 57.0 mmol) and 6-(difluoromethoxy)-2,5-dimethylpyridin-3-amine (10.7g, 57.0 mmol) were combined under N₂ in a 2 L, 3-neck, round-bottomedflask equipped with a thermometer and an addition funnel. THF (570 mL)was added and the mixture was cooled to 0° C. NaHMDS (119.7 mL, 119.7mmol, 1 M in THF) was added dropwise via addition funnel over 20 min ata rate to maintain the internal temperature below 5° C. After theaddition was complete, the reaction mixture was stirred at 0° C. for anadditional 15 min. The reaction was quenched by the addition ofsaturated aqueous NH₄Cl (60 mL). The mixture was transferred to aseparatory funnel containing water (400 mL) and the aqueous layer wasextracted with ether (3×300 mL). The combined organic layers were washedwith brine, dried over MgSO₄, filtered and concentrated. The product waspurified by column chromatography on silica gel (30% ethyl acetate inhexanes) to afford(S)-5-chloro-1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]pyrazin-2(1H)-one(22.1 g, 94% yield) as a pale yellow solid which was subsequentlyrecrystallized from heptane to furnish colorless needles: mp103.4-104.4° C.; [α]²⁵D−41.9 (c 0.807, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ8.42 (s, 1H), 8.02 (s, 1H), 7.46 (t, J=74.0 Hz, 1H), 6.96 (s, 1H),4.19-4.14 (m, 1H), 3.75 (dd, J_(AB)=10.3, J_(AX)=6.3 Hz, 1H), 3.67 (dd,J_(BA)=10.3, J_(BX)=3.5 Hz, 1H), 3.34 (s, 3H), 2.44 (s, 3H), 2.26 (s,3H), 1.32-1.26 (m, 1H), 0.81-0.74 (m, 1H), 0.63-0.54 (m, 1H), 0.52-0.47(m, 1H), 0.36-0.29 (m, 1H); HRMS (ESI) m/e 415.1360 [(M+H)⁺, calcd forC₁₈H₂₂N₄O₃ClF₂ 415.1349]. Anal. Calcd for C₁₈H₂₁N₄O₃ClF₂: C, 52.11; H,5.10; N, 13.50. Found: C, 52.05; H, 4.99; N, 13.48.

(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-[6-((difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile.(S)-5-Chloro-1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluoromethoxy)-2,5-dimethyl-pyridin-3-ylamino]pyrazin-2(1H)-one(99.3 mg, 0.24 mmol) was dissolved in anhydrous NMP (3 mL). To thissolution was added Pd(dppf)Cl₂, CH₂Cl₂ (78.4 mg, 0.096 mmol), Zn(CN)₂(60 mg, 0.51 mmol) and Zn powder (24 mg, 0.37 mmol). N₂ was bubbledthrough the reaction mixture for 15 min to expel the dissolved O₂ andthe reaction mixture was maintained at 120° C. under Argon for 1 h.LC-MS analysis indicated a 92.5% conversion to required product. LRMS(ESI) m/e 406.3 [(M+H)⁺, calcd for C₁₉H₂₂N₅O₃F₂ 406.2].

(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-methoxy-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile.(S)-5-Chloro-1-(cyclopropyl-2-methoxyethyl)-3-[6-(methoxy)-2,5-dimethyl-pyridin-3-ylamino]pyrazin-2(1H)-one(3.00 g, 7.93 mmol) was dissolved in 63 mL of N-methylpyrrolidinone.Pd(dppf)Cl₂.CH₂Cl₂ (648 mg, 0.793 mmol), Zn(CN)₂ (1.86 g, 15.9 mmol) andZn powder (619 mg, 9.52 mmol) were added to the solution. After bubblingN₂ through the solution for 15 min, the dark mixture was maintainedunder argon at 120° C. for 23 h. LC-MS analysis of the reaction mixturerevealed a 98.6% conversion. The reaction mixture was cooled to ambienttemperature, filtered through a short bed of Celite and the Celite cakewas washed with NMP. The filtrate was evaporated in vacuo (bathtemperature 60° C.). The dark residue was partitioned between EtOAc (350mL) and H₂O (60 mL). The organic layer was washed with water (2×50 mL)and brine (50 mL), dried over Na₂SO₄, and then evaporated in vacuo. Thedark brown residue was then purified by column chromatography on silicagel (30% ethyl acetate in hexanes, R_(f)=0.3) to give pure(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-methoxy-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile(2.83 g, 96.5%) as pale yellow solid: ¹H NMR (CDCl₃) δ 7.86 (s, 1H),7.49 (s, 1H), 4.16-4.13 (m, 1H), 3.94 (s, 1H), 3.74 (dd, 1H, J_(AB)=10.4Hz, J_(AX)=5.2 Hz), 3.66 (dd, J_(BX)=3.2 Hz), 3.35 (s, 3H), 2.43 (s,3H), 2.19 (s, 3H), 1.39-1.32 (m, 1H), 0.85-0.79 (m, 1H), 0.67-0.61 (m,1H), 0.55-0.50 (m, 1H), 0.34-0.29 (m, 1H); ¹³C NMR (CDCl₃): 158.7,151.4, 148.1, 144.1, 133.1, 125.7, 124.7, 118.4, 117.3, 107.8, 77.4,77.1, 76.9, 72.7, 62.3, 59.5, 53.6, 20.0, 15.8, 11.2, 6.2, 4.1; LRMS(ESI) m/e 370 [(M+H)⁺, calcd for C₁₉H₂₄N₅O₃ 370]; t_(R)=2.0 min (SolventA: MeOH:H₂O:TFA=10:90:0.1; Solvent B; MeOH:H₂O:TFA=90:10:0.1; 40% B in Ato 100% B in A linear gradient in a 3 min run with 1 min hold time atthe end, λ=280 nm).

(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-hydroxy-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile.(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-methoxy-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile(3.00 g, 8.12 mmol) and KI (6.2 g, 37.3 mmol) in glacial acetic acid (80mL) was heated at 100° C. with stirring for 1 h. LC-MS analysisindicated>98% conversion to the required product. The reaction mixturewas cooled to ambient temperature and acetic acid was evaporated invacuo. The residue was partitioned between ethyl acetate (3×150 mL) andH₂O (˜100 mL). The organic layer was washed with brine (100 mL) and thendried over Na₂SO₄. The organic layer was evaporated in vacuo and thedark brown residue was purified by Biotage SiO₂ column chromatography(5% MeOH/CH₂Cl₂, R_(f)=0.16) to give pure(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-hydroxy-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile(2.33 g, 81%) as pale yellow solid: ¹H NMR (CDCl₃) δ 7.85-7.55 (br. s,1H), 7.48 (s, 1H), 4.16-4.12 (m, 1H), 3.73 (dd, 1H, J_(AB)=10.3 Hz,J_(AX)=4.9 Hz), 3.66 (dd, J_(BX)=2.2 Hz), 3.35 (s, 3H), 2.40-2.20 (br s,6H), 1.39-1.32 (m, 1H), 0.85-0.79 (m, 1H), 0.67-0.61 (m, 1H), 0.55-0.51(m, 1H), 0.34-0.29 (m, 1H); LRMS (ESI) m/e 356 [(M+H)⁺, calcd forC₁₈H₂₁N₅O₃ 356]; t_(R=)1.8 min (Solvent A: MeOH:H₂O:TFA=10:90:0.1;Solvent B: MeOH:H₂O:TFA=90:10:0.1; 0% B in A to 100% B in A lineargradient in a 3 min run with 1 min hold time at the end, λ=280 nm).

(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile.A mixture of pure(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-hydroxy-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile(2.3 g, 6.5 mmol) and CsF (395 mg, 2.6 mmol) was added to acetonitrile(65 mL). To the resulting suspension, trimethylsilyl2,2-difluoro-2-(fluorosulfonyl)acetate (3.82 mL, 19.4 mmol) was slowlyadded (use extreme caution! The reagent can cause dermatological damageand should always be handled in hood). LC-MS analysis after 1 h showedthe reaction to be complete (89% conversion to required product),Volatiles were evaporated in vacuo and the residue was partitionedbetween EtOAc (3×120 mL) and H₂O (˜100 mL). The organic layer was washedwith water (2×50 mL) and brine (50 mL), and dried over Na₂SO₄. The ethylacetate was evaporated in vacuo and the residue was dissolved in DMF (10mL). The solution was applied to Ca₈ silica gel column (14 cm×5 cm)equilibrated with MeCN:H₂O (1:2). The column was eluted with 33%MeCN/H₂O (500 mL), 45% MeCN/H₂O (500 mL), 60% MeCN/H₂O (1000 mL), 70%MeCN/H₂O (500 mL). Individual fractions were analyzed by LC-MS (roductcomes out in between 60%-70% MeCN/H₂O). Fractions containing pureproduct were combined and concentrated in vacuo to give pure(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile(2.28 g, 87% yield) as pale yellow solid: ¹H NMR (CDCl₃) δ 8.43 (s, 1H);8.01 (s, 1H); 7.56 (s, 1H); 7.49 (t, J_(H-F)=73.8 Hz, 1H); 4.15-4.18 (m,1H); 3.76 (dd, J_(AB)=10.4, J_(AX)=5.2 Hz, 1H); 3.68 (dd, J_(BA)=10.4,J_(BX)=3.1 Hz, 1H); 3.37 (s, 3H); 2.46 (s, 3H); 2.30 (s, 3H); 1.35-1.42(m, 1H); 0.82-0.88 (m, 1H); 0.64-0.69 (m, 1H); 0.53-0.58 (m, 1H);0.31-0.36 (m, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 153.0, 151.5, 147.8,143.8, 133.3, 129.4, 125.6, 119.1, 117.2, 114.8 (t, J=256.1 Hz, 1 C),107.6, 72.7, 62.6, 59.6, 20.0, 15.5, 11.4, 6.4, 4.2; ⁹F NMR (CDCl₃)δ-88.73 (d); LRMS (ESI) m/e 406 [(M+H)⁺, calcd for C₁₉H₂₂N₅O₃F₂ 406];t_(R)=2.7 min (Solvent A: MeOH:H2O:TFA=10:90:0.1; Solvent B:MeOH:H₂O:TFA =90:10:0.1; 40% B in A to 100% B in A linear gradient in a3 min run with 1 min hold time at the end, λ=280 nm).

It will be evident to one skilled in the art that the present disclosureis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

1. The compound(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile,or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising a therapeutically effective amount of(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrileand a pharmaceutically acceptable adjuvant, carrier or diluent.
 3. Amethod for the treatment of psychiatric or neurological conditionsassociated with CRF which comprises administering a therapeuticallyeffective amount of(S)-4-(1-cyclopropyl-2-methoxyethyl)-6-(6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrileto a patient.
 4. The method of claim 3 where the condition isdepression.
 5. The method of claim 3 where the condition is anxiety oran anxiety related disorder.
 6. The method of claim 3 where thecondition is irritable bowel syndrome.
 7. The method of claim 3 wherethe condition is addiction or negative aspects of drug and alcoholwithdrawal.